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CIRCULAR DICHROISM
Introduction
Circular dichroism (CD) spectroscopy is a type of absorption spectroscopy that can provide
information on the structures of many types of biological macromolecules. Biological
macromolecules such as proteins and DNA are composed of optically active (chiral) elements and
because they can adopt different types of three-dimensional structures, each type of molecule
produces a distinct CD spectra (figure 1).
Figure 1. CD spectra of triosephosphate isomerase (helices (H) : 0.52, beta-sheets (S) : 0.14,
turns (T) : 0.11, other structure types (O) : 0.23), hen egg lysozyme (H:0.36, S:0.09, T:0.32,
O:0.23), myoglobin (H:0.78, S:0.0, T:0.12, O:0.10), and chymotrypsin (H:0.10, S:0.34, T:0.20,
O:0.36).
Theory
Recall that electromagnetic radiation is a complex wave form that can be considered to behave as
two simple wave motions at right angles to each other (figure 2).
Figure 2. Electromagnetic wave consisting of a sinusoidal oscillations of an electric field and a
magnetic field.
One of these waves is magnetic (M, green) and the other is electric (E, blue). Electromagnetic
waves are generated by oscillating electric or magnetic dipoles and are propagated at the speed of
light (c). Since the E- and the M-components are always perpendicular to each other, it is sufficient,
in many cases, to consider only the E-component in describing the wave.
Although the amplitude of the E-wave oscillates in the zx-plane in the figure above, it could
oscillate in any direction perpendicular to the direction of propagation (z). Unpolarized light, the
type we get from the sun or a light bulb contains oscillations of the E - components in all directions
perpendicular to the direction of propagation. Linearly polarized light results when the direction of
the E-component is restricted to a plane perpendicular to the direction of propagation while its
magnitude oscillates. Circularly polarized light is another form of polarization - in this case, the
magnitude of the oscillation is constant and the direction oscillates (figure 3).
Figure 3. Schematic diagram showing the electronic component of linearly polarized light
(left) and right-handed circularly polarized light (right). Below each are the electronic
component vectors as viewed along the axis of propagation from left of the diagram above.
The differential absorption of radiation polarized in two directions as function of frequency is called
dichroism. When applied to plane polarized light, this is called linear dichroism; for circularly
polarized light, circular dichroism. Chiral or asymmetric molecules produce a CD spectrum
because they absorb left and right handed polarised light to different extents and thus are considered
to be "optically active".
The wavelengths of light that are most useful for examining the structures of proteins and DNA are
in the ultraviolet (UV) or vacuum ultraviolet (VUV) ranges (from 160 to 300 nm) because these are
the regions of the electronic transitions of the peptide backbone and side chains (histidine, cysteine,
tryptophan, tyrosine and phenylalanine) in proteins and the purine and pyrimidine bases in DNA.
Specifically, protein secondary and tertiary structures can be monitored by the peptide transitions in
the far UV (~190-220 nm) and by the aromatic side chain transitions in the near UV (~270-290
nm), respectively. Other chromophores in the visible region (for coloured proteins) may be valuable
for establishing the fidelity of tertiary and quaternary folding. CD spectra can then be analyzed for
the different secondary structural types: alpha helix, parallel and antiparallel beta sheet, turn, and
other (figure 4).
Figure 4. Circular dichroism spectra of "pure" secondary structures. Redrawn from Brahms
& Brahms, 1980.
Applications
CD has an important role in the structural determinants of proteins. However, the effort expended in
determining secondary structure elements is usually not worth it because it is somewhat unreliable.
The real power of CD is in the analysis of structural and conformational changes in a protein
upon some perturbation (salt, pH, temperature). These changes can be quantitated by CD since
shifts in conformation involving as few as 10 amino acids may be readily detectable.
It can also be efficiently used in comparison of the structure of an engineered protein to the
parent protein. For example, it is required that the overall structure of a mutated protein hasn’t
changed considerably compared to the wild type protein. Previously, a hydrophobic dyes (for
example Nile Red) was used to detect changes, but its use was limited due to the uncertainty of the
results. Also, the the changes had to be extremely drastic (a hydrophobic core displayed on the
outside of a protein). CD spectroscopy can therefore elucidate whether a change of a single amino
acid has caused any slighter perturbations on the overall secondary structure topology. The effect
would be similar as in protein denaturation, the studying of which CD spectroscopy fits well, too
(figure 5).
Figure 5. Protein denaturation – renaturation equilibrium
CD can also be used in an empirical manner to determine protein unfolding, ligand or drug
binding, etc. CD is rapid and can be used to analyze a number of candidate proteins from which
interesting candidates can be selected for more detailed structural analysis like NMR or X-ray
crystallography. In advance of obtaining a crystal structure, CD can be useful in conjunction with
modelling efforts: it can provide a good test of the secondary structural content of any model
produced, and can be used to determine the class of molecule. For crystallography, it may be used
as an assay of whether a particular structure will be a reasonable model for molecular replacement.